Biology subjects

Sirohiwal, A.

Publications and source records attributed to Sirohiwal, A..

3 recordsLinked to original sources

bioRxiv2024

Modified Chlorophyll Pigment at ChlD1 Tunes Photosystem II Beyond the Red-Light Limit

Photosystem II (PSII) is powered by the light-capturing properties of chlorophyll a pigments that define the spectral range of oxygenic photosynthesis. Some photosynthetic cyanobacteria can acclimate to growth in longer wavelength light by replacing five chlorophylls for long wavelength pigments in specific locations, including one in the reaction center (RC). However, the exact location and the nature of this long wavelength pigment still remain uncertain. Here we have addressed the color-tuning mechanism of the farred light PSII (FRL-PSII) by excited state calculations at both the ab initio correlated (ADC2) and linear-response time-dependent density functional theory (LR-TDDFT) levels in combination with large-scale hybrid quantum/classical (QM/MM) simulations and atomistic molecular dynamics. We show that substitution of a single chlorophyll pigment (ChlD1) at the RC by chlorophyll d leads to a spectral shift beyond the far-red light limit, as a result of the protein electrostatic, polarization and electronic coupling effects that reproduce key structural and spectroscopic observations. Pigment substitution at the ChlD1 site further results in a low site energy within the RC that could function as a sink for the excitation energy and initiate the primary charge separation reaction, driving the water oxidation. Our findings provide a basis for understanding color-tuning mechanisms and bioenergetic principles of oxygenic photosynthesis at the far-red light limit.

biophysics↗

bioRxiv2024

Mechanistic principles of hydrogen evolution in the membrane-bound hydrogenase

The membrane-bound hydrogenase (Mbh) from Pyrococcus furiosus is an archaeal member of the Complex I superfamily. It catalyzes the reduction of protons to H2 gas powered by a [NiFe] active site and transduces the free energy into proton pumping and Na+/H+-exchange across the membrane. Despite recent structural advances (1-4), the mechanistic principles of H2 catalysis and ion transport in Mbh remain elusive. Here we probe how the redox chemistry drives the proton reduction to H2 and how the catalysis couples to conformational dynamics in the membrane domain of Mbh. By combining large-scale quantum chemical density functional theory (DFT) and correlated ab initio wave function methods with atomistic molecular dynamics simulations, we show that the proton transfer reactions required for the catalysis are gated by electric field effects that direct the protons by water-mediated reactions from Glu21L towards the [NiFe] site, or alternatively along the nearby His75L pathway that also becomes energetically feasible in certain reaction steps. These local proton-coupled electron transfer (PCET) reactions induce conformational changes around the active site that provide a key coupling element via conserved loop structures to the ion transport activity. We find that H2 forms in a heterolytic proton reduction step, with spin crossovers tuning the energetics along key reaction steps. On a general level, our work showcases the role of electric fields in enzyme catalysis, and how these effects are employed by the [NiFe] active site of Mbh to drive the PCET reactions and ion transport. Significance statementHydrogen (H2) serves as a crucial solar fuel in renewable energy systems that can be efficiently produced by microbial hydrogenases. Here we probe the elusive mechanistic principles underlying the H2 production in the ancient membrane-bound hydrogenase (Mbh) from the thermophilic archaeon Pyrococcus furiosus. Distinct from other hydrogenases, Mbh not only produces H2, but it couples this activity with ion transport across a membrane that powers the archaeal energy metabolism. Our study elucidates key mechanistic principles underlying H2 production and shed light on energy transducing enzymes that led to the evolution of modern mitochondrial respiratory enzymes.

biochemistry↗

bioRxiv2023

Pigment Binding in The Light-Dependent Protochlorophyllide Oxidoreductase

The Light-Dependent Protochlorophyllide Oxidoreductase (LPOR) is a key enzyme in chlorophyll biosynthesis and its photocatalytic mechanism has long intrigued researchers. However, the lack of structural data for the active complex has impeded understanding of its reaction mechanism. A recent high-resolution structure of enzyme in the active conformation has established a robust foundation for validating hypotheses concerning pigment binding, residue involvement, and consequently, the reaction mechanism. Surprisingly, this new structure challenges previously proposed mechanisms, especially concerning the orientation of the bound protochlorophyllide (Pchlide) pigment. In this study, we employ molecular dynamics and hybrid quantum-mechanics/molecular-mechanics (QM/MM) simulations along with site-directed mutagenesis to compare two Pchlide binding modes: one aligned with previous proposals (mode A), and the other consistent with the recent experimental data (mode B). Binding energy calculations reveal thermodynamic instability of binding mode A due to nonspecific interactions, while mode B exhibits distinct stabilizing interactions yielding favorable binding. QM/MM-based local energy decomposition analysis unravels a complex interaction network that reinforces pigment stabilization in this conformation. Notably, interactions involving Tyr177, His319, and the carboxyl group at C131 influence the pigments excited state energy and potentially contributing to the substrate specificity of the enzyme. Our results uniformly favor binding mode B as represented in the new cryo-EM structure, over the previously assumed mode A. These findings challenge established interpretations and underscore the need for a comprehensive re-evaluation of the reaction mechanism of LPOR that correctly considers pigment interactions and substrate orientation in the binding pocket. Significance StatementA crucial step in the biosynthesis of the all-important photosynthetic pigment chlorophyll is the reduction of a double C=C bond in its precursor protochlorophyllide (PChlide). This is catalyzed by the Light-Dependent Protochlorophyllide Oxidoreductase (LPOR) via an extremely rare example of a biological photocatalytic reaction. Understanding of the LPOR mechanism has been hindered by limited insight into the structure of its active complex. A recent high-resolution LPOR cryo-EM structure substantiates pigment binding, residue interactions, and the reaction mechanism, but contrasts markedly with all previous assumptions regarding the binding mode of the substrate PChlide. Using molecular dynamics simulations, quantum-mechanics/molecular-mechanics calculations, and mutagenesis, we compare and evaluate the two possible Pchlide binding modes, the one assumed previously (mode A) and the one supported by recent data (mode B). Our findings conclusively favor mode B, challenging prior assumptions and pointing toward novel mechanistic possibilities for this unique photocatalytic reaction.

biochemistry↗